nutrients

Article Microencapsulation and the Characterization of Polyherbal Formulation (PHF) Rich in Natural Polyphenolic Compounds

Syed Ammar Hussain 1,† ID , Ahsan Hameed 1,† ID , Yusuf Nazir 1 ID , Tahira Naz 1 ID , Yang Wu 1, Hafiz Ansar Rasul Suleria 2,3,4,* ID and Yuanda Song 1,*

1 Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, Zibo 255049, China; [email protected] (S.A.H); [email protected] (A.H.); [email protected] (Y.N.); [email protected] (T.N); [email protected] (Y.W.) 2 UQ Diamantina Institute, Translational Research Institute, Faculty of Medicine, The University of Queensland, 37 Kent Street Woolloongabba, Brisbane, QLD 4102, Australia 3 Department of Food, Nutrition, Dietetics and Health, Kansas State University, Manhattan, KS 66506, USA 4 Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Pigdons Road, Waurn Ponds, VIC 3216, Australia * Correspondence: hafi[email protected] (H.A.R.S.); [email protected] (Y.S.); Tel.: +61-470-439-670 (H.A.R.S.); +86-139-0617-4047 (Y.S.) † These authors contributed equally to this work.



Received: 22 May 2018; Accepted: 22 June 2018; Published: 28 June 2018


Abstract: Microencapsulation of polyherbal formulation (PHF) extract was carried out by freeze drying method, by employing gum arabic (GA), gelatin (GE), and maltodextrin (MD) with their designated different combinations as encapsulating wall materials. Antioxidant components (i.e., total phenolic contents (TPC), total flavonoids contents (TFC), and total condensed tannins (TCT)), antioxidant activity (i.e., DPPH, β-carotene & ABTS+ assays), moisture contents, water activity (aw), solubility, hygroscopicity, glass transition temperature (Tg), particle size, morphology, in vitroα-amylase and α-glucosidase inhibition and bioavailability ratios of the powders were investigated. Amongst all encapsulated products, TB (5% GA & 5% MD) and TC (10% GA) have proven to be the best treatments with respect to the highest preservation of antioxidant components. These treatments also exhibited higher antioxidant potential by DPPH and β-carotene assays and noteworthy for an ABTS+ assays. Moreover, the aforesaid treatments also demonstrated lower moisture content, aw, particle size and higher solubility, hygroscopicity and glass transition temperature (Tg). All freeze dried samples showed irregular (asymmetrical) microcrystalline structures. Furthermore, TB and TC also illustrated the highest in vitro anti-diabetic potential due to great potency for inhibiting α-amylase and α-glucosidase activities. In the perspective of bioavailability, TA,TB and TC demonstrated the excellent bioavailability ratios (%). Furthermore, the photochemical profiling of ethanolic extract of PHF was also revealed to find out the bioactive compounds.

Keywords: microencapsulation; polyphenols; freeze-drying; antioxidant activity; in vitro dialyzability; in vitro anti-diabetic potential

1. Introduction In the past decade, research has been focused in exploring naturally occurring antioxidants to circumvent the multifaceted health related complexities arising due to overproduction of reactive

Nutrients 2018, 10, 843; doi:10.3390/nu10070843 www.mdpi.com/journal/nutrients Nutrients 2018, 10, 843 2 of 25 oxygen species (ROSs) in body [1,2]. The excess production of these ROSs are considered serious issue for human health as their surplus generation can lead to different patho-physiological conditions like fast aging process via damaging the nucleic acids and changing in the conformation of proteins, heart-related disorders (i.e., cardiovascular disorders (CVDs)), diverse types of cancers, immunity related dysfunctions, inflammation, membranous lipid oxidation, decline of hydroperoxide synthesis, neurodegenerative disorders, lungs and kidney illness, UV-irradiation, and osteoporosis/bone-related diseases and health-related disease called “oxidative stress” [3]. In addition, direct correlation between oxidative stress and insulin resistance (key factor for type-II DM) has also been elaborated in mini review by Hurrle et al. [4]. Amongst naturally-occurring antioxidants, polyphenols and their derivative compounds represented a diverse class of ubiquitous material i.e., from simple molecules to complex configuration such as phenolic acids; hydroxybenzoic and hydroxycinnamic acids, hydrolyzable and condensed tannins, and flavonoids, these are most important compounds for nutraceutical, therapeutics and pharmacological point of view [5,6] and revealed various health endorsing activities: antioxidant activity (i.e., free radicals scavenging, declining of hydroperoxide development, hampering the lipid oxidation), anti-diabetic, anti-malarial, and anticancer activity etc. [5,7]. In the prospective of natural polyphenols, polyherbal formulations are considered as a great source all over the globe due to their dynamic medicinal and therapeutic claims. Moreover, previous investigations illustrated that selected individual plants contained abundant quantity of polyphenols and their herbal combinations were found to produce best antioxidant activity among all individual extracts due to synergistic effect. Synergism played a vital role via two different kind of mechanism in context of interaction i.e., pharmacokinetic (PK) and pharmacodynamics (PD) [8,9]. Owing to synergism, polyherbal formulation demonstrated vast advantages over single herbal formulation (SHF) likewise: superior restorative effect can be attained with a PHF; to acquire enviable pharmacological accomplishment low dosage would be required, consequently lessening the risk of harmful side effects. Additionally, PHF facilitate the patient’s convenience by eradicating the need of taking more than one formulation at a time, which ultimately leads to better compliance and therapeutic effect. All the aforesaid advantages have outcome in the attractiveness of PHF in the marketplace when compare to SHF [10]. Polyphenols are incredibly sensitive in diverse range of circumstances, during food processing and storage practice likewise; high temperature of surrounding, incidence of oxygen and light, pH, existence of oxidative enzymes, moisture contents [11]. The degradation of natural antioxidants may hamper the possible effectiveness of application of these antioxidants in food/nutraceutical and pharmaceutical applications and commercially available anti-diabetic drugs also produce unconstructive effects on other metabolisms [12], so supplementation of anti-hyperglycemic substances, which also possess antioxidant properties, might be an alternative therapy to overcome this critical condition [13,14]. To address these shortcomings and to augment the antioxidant stability and preserve their diverse bioactivities including anti-inflammatory, anti-cancer, anti-microbial, anti-diabetic capabilities, the microencapsulation has been employed successfully as a reliable technique to circumvent the unwanted degradation of bioactive compounds, shielding them from adverse environmental circumstances. Furthermore, various type of wall material has been used for microencapsulation procedure, but cost effectiveness and physico-chemical distinctiveness must be considered, including: hygroscopicity, biodegradability, emulsifying feature, adaptability to gastrointestinal tract (GT), viscosity, solids content [15]. At present, the preferred wall materials for microencapsulation for various fruit juices and plant/herbs extracts are maltodextrins (MD), gum arabic (GA) and gelatin (GE) [16]. Maltodextrin of various dextrose equivalents (DE) are generally used as wall material owing to their distinct characteristics likewise; low viscosity, high solubility in water and their solutions are monochromic in appearances. These features made them frequently used carrier/wall materials in the micro-encapsulation procedure. Gum arabic (exudates of acacia), owing to its unique features Nutrients 2018, 10, 843 3 of 25 i.e., naturally colorless, low viscosity, high retention of volatiles and ability to make stable emulsion is ultimately considered as excellent encapsulating agent whereas its high economic cost provoked researcher for full or partial replacement of the encapsulation agent [16–18]. In addition, gelatin is also a better option for microencapsulation because of its superior characteristics for emulsification, film-formation, water solubility, last but not least ability to form finer dense complex. According to Fang and Bhandari [19], a sole microencapsulating agent has limitation over all required attributes to improve microencapsulation effectiveness, eventually has been resolved by using different combination of polymers due to their diverse features. The selection for polymer’s combinations which possibly consequence in superior microencapsulating efficiency and regarded economically suitable than the single biopolymers has been becoming the point of emerging interest [19,20]. In the current study, polyherbal formulation was firstly made with equal ratio of roots of Chlorophytum borivilianum, roots of Astragalus membranaceus, roots of Eurycoma longifolia, and seeds of Hygrophila spinosa T. Anders having previously proven diverse ethno-pharmacological applications [21–24] as polyphenols enriched nutrient supplement, then PHF extract was further microencapsulated by freeze drying method using different wall materials, subsequently antioxidant components (i.e., TPC, TFC, and TCT), antioxidant activity (i.e., DPPH, β-carotene & ABTS+ assays), anti-diabetic potential (i.e., in vitro α-amylase and α-glucosidase inhibition), physical properties like; moisture contents, water activity (aw), solubility, hygroscopicity, glass transition temperature (Tg), morphological characteristics (i.e., particle size, morphology), and bioavailability ratios of the microencapsulated powders were investigated. In last, the chemo-profiling for ethanolic extract of PHF was also studied.

2. Materials and Methods

2.1. Materials, Chemicals, Reagents and Encapsulating Agents All different parts of herbs (detail in Section 2.2) were purchased from Faisalabad-Pakistan and their identification and respective characteristics were authenticated by Prof. M. Jafar Jaskani from Institute of Horticulture, University of Agriculture Faisalabad (UAF) Pakistan. All chemicals used were of analytical grade or higher where suitable. DPPH (2,2-diphenyl-1-picryl-hydrazyl), Folin-Ciocalteu (FC),β-carotene, Butylated hydroxyltoluene (BHT), TWEEN 20, quercetin, Sodium carbonate,ABTS (2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid), α-tocopherol, Linoleic acid, (+)-catachin, quercetin, AlCl3·6H2O, HCl, Vanilline, NaOH, Potassium persulfate,Trolox, gallic acid were purchased from Sigma-Aldrich GmbH (Sternheim, Germany). α-amylase fromporcine pancreas,α-glucosidase from Saccharomyces cerevisiae, paranitrophenyl-glucopyranoside, pepsin (porcine-7000), bile salts pancreatin (p-1750), piperazine-NN-bis (2-ethane-sulfonic acid) di-sodium salt (PIPES), gelatin (GE), HPLC-grade methanol, acetonitrile ethanol, acetone were supplied by Sigma-Aldrich (St. Louis, MO, USA), soluble starch (extra pure) was obtained from J. T. Baker Inc. (Phillipsburg, NJ, USA). Ultra-pure water (18 MΩ cm−1) was acquired from Milli–Q purification device (Millipore Co., Billerica, MA, USA). Sodium hydrogen carbonate was purchased from Merck (Darmstadt, Germany). Sea sand was of 200–300 grain size from Scharlau (Barcelona, Spain). The encapsulating agents were: gum arabic (Sangon Biotech Co., Shanghai, China), maltodextrin (Dextrose equivalent of 12) was purchased from Corn Products (Cabo de Santo Agostinho, Pernambuco, Brazil).

2.2. Polyherbal Formulation Polyherbal formulation was made by combining the root of Chlorophytum borivilianum, roots of Astragalus membranaceus, roots of Eurycoma longifolia, and seeds of Hygrophila spinosa T. Anders, in a ratio of 1:1:1:1 respectively. Nutrients 2018, 10, 843 4 of 25

2.3. Preparation of Sample Firstly, the roots and seeds of aforesaid herbs were cut into small pieces, followed by thorough washing with deionized water in order to avoid any contamination. The PHF material was then dried for 12 days in dark in well ventilated room at room temperature (23 ± 8 ◦C), and subsequently grounded with mortar and pestle to make crude powder with the help of liquid nitrogen, until a uniform sieve size equivalent to (1.0 mm) was achieved. The resulting powder was stored at −80 ◦C in inert vacuum bags until used for extraction as followed.

2.4. Pressurized Liquid Extraction (PLEx) PLEx was executed in a Dionex ASE 350 system (Dionex, Sunnyvale, CA, USA) with the powder of PHF obtained as mentioned above. Aliquot of 5.0 g of powder of PHF was mixed with diatomaceous earth (1/1) and placed in a 34 mL stainless–steel cells. The extraction was performed via 3 consecutively applied steps with absolute solvents of increasing polarity, in order to get the maximum possible number and amount of secondary metabolites of various polarities and miscibilities, namely, acetone, ethanol, methanol and their aqueous mixtures with water (1:10, 3:10), and pure water. Extraction time was of 22 min; pressure 10.6 MPa; temperature 75 ◦C (for acetone, ethanol and methanol) and 135 ◦C (for water). Organic solvents were removed in a rotary vacuum evaporator at 38 ◦C, while the residual water was removed in a freeze drying unit. The extracts after solvent evaporation were placed under nitrogen flow for 20 min and stored in dark glass bottles at −80 ◦C until analyzed.

2.5. Development of Microencapsulated Powder Products In order to prepare the particular dispersions, 100 mL of water was mixed with aforesaid PHF extract individually with different preselected combination of microencapsulating wall materials as follow: A (5% GA & 5% GE) (hereafter referred and discussed as TA); B (5% GA & 5% MD) (hereafter referred and discussed as TB), C (10% of GA)(hereafter referred and discussed as TC), and D (10% ◦ of MD) (hereafter referred and discussed as TD),under constant shaking with 220 rpm, at 35 C for 30 min by a shaking unit (CIMO instrument manufacturing Co., Shanghai, China). Afterward, these dispersions were microencapsulated through lyophilization process for formulating four distinctive treatments. i.e., TA,TB,TC,TD. For microencapsulation by means of freeze-drying process, the above-mentioned dispersions/ emulsions were kept at −20 ◦C (freezer) for 48 h. Subsequently, the samples were placed in lyophilization unit (Labconco, Kansas, MO, USA) for freeze drying at −56.5 ◦C, with vacuum pressure of 4.61 mmHg for 60 h. After the completion of freeze drying process, the samples were crushed utilizing a mortar and pestle assembly. Finally, the desirable final microencapsulated products were sealed in polyethylene bags and aluminum pouches as well and stored in desiccator encompassing silica until further analysis.

2.6. Determination of Bioactive Compounds and Their Bioactivities after Microencapsulation Bioactive components which were determined after the microencapsulation were total phenolic compounds (TPC), total flavonoids compounds (TFC), total condensed tannins (TCT). While the bioactivities of the microencapsulated powders were measured in terms of total antioxidant activity determined by β-carotene bleaching assay (TOAA), ABTS+ radical scavenging activity, and DPPH scavenging capacity. All these spectrophotometric analysis were performed according to previously developed methods with minor alteration [2,25,26]. The results for ABTS+ radical scavenging activity are deliberated as EC50 values (mg of extract/mL) for comparison. Effectiveness of antioxidant properties is inversely correlated with EC50 value. Nutrients 2018, 10, 843 5 of 25

2.7. Determination of the Physical Properties of the Microencapsulated Powders

2.7.1. Moisture Content The moisture contents of the microencapsulated products were estimated by using the method describes in manual AOAC [27], i.e., by calculating the loss of sample after weight after heat up at 105 ◦C.

2.7.2. Water Activity (aw)

The water activity (aw) of all lyophilized samples was calculated through the direct analysis in electronic meter (Aqualab 3TE-Decagon, Pullman, WA, USA), to gain the constant state the samples were firstly placed at 25 ◦C for at least 15 min.

2.7.3. Solubility The solubility of microencapsulated products was measured by the method described by Cano-Chauca et al. [28], with minute alterations. The sample’s quantity of 1.0 g was mixed up with 100 mL distilled water in beaker and stirred with magnetic stirrer (MS-H-S10) for 20 min. After that the centrifugation of solution carried out at 3000× g (Thermo Scientific, Waltham, MA, USA) for 10 min. The quantity of 25 mL of the supernatant was transferred to a petriplates (pre-weighted) and dried in oven at 105 ◦C for 4.0 h. The solubility was measured as a result of weight difference and demonstrated in the term of percentage (%).

2.7.4. Hygroscopicity For the estimation of hygroscopicity, the microencapsulated powders of 1.0 g were placed in dessicator with saturated NaCl solution (74.6%) at temperature of 25 ◦C. After 1 week, samples were weighed and hygroscopicity were calculated in the term of percentage (%) [29].

2.7.5. Glass Transition Temperature (Tg)

The glass transition temperature (Tg) of the microencapsulated products was calculated by means of differential scanning calorimetry (DSC) (DSC-2000-New Castle, DE, USA). The weight of 7–8 mg of sample was placed in aluminum hermetic pots. For the reference purpose, an aluminum pan without sample was used. Ultra-pure nitrogen N2 was used as purge gas (flow rate 50 mL/min). The temperature ranged from −80 ◦C to 120 ◦C at a heating rate of 40 ◦C/min. The glass transition temperature was determined by utilizing software of TA Universal Analysis 2000.

2.8. Morphology and Size Distribution The configuration of micro-particles obtained from diverse encapsulating wall material and their combinations were examined by scanning electron microscope (Quanta 250 EFI). At first, very minute was fixed on surface of double sided tape of carbon then finally evaluated the samples under microscope with 400× magnification. The analysis for particle size distribution average and particle size was conducted by the means of ImageJ (NIH, Bethesda, MD, USA).

2.9. In Vitro Assays

2.9.1. α-Amylase Inhibition Assay The inhibition of α-amylase was determined using an assay modified from the Worthington Enzyme Manual [30]. Aliquot 0–4 mg/mL in DMSO (v/v 1:1) of each encapsulated PHF samples was prepared and 500 µL of each sample were mixed with 500 µL of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.5 mg/mL) and incubated at 25 ◦C for 10 min. After pre-incubation, 500 µL of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was Nutrients 2018, 10, 843 6 of 25 added to each tube at timed intervals. The reaction mixtures were then incubated at 25 ◦C for 10 min. The reaction was stopped with 1.0 mL of dinitrosalicylic acid color reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted by adding 15 mL of distilled water, and the absorbance was measured at 540 nm using a micro-plate reader (Thermomax, Molecular Device Co., Sunnyvale, CA, USA). The experiments were performed in duplicate and the absorbance of sample blanks (buffer instead of enzyme solution) and a control (buffer in place of sample extract) were also recorded. The absorbance of the final each encapsulated PHF sample was obtained by subtracting its corresponding sample blank reading. Acarbose was prepared in distilled water and used as positive controls. The percentage inhibition was calculated using the formula;

% Inhibition = {(Ac − Ae)/Ac} 100 where Ac and Ae are the absorbance of the control and extract, respectively. IC50 values (inhibitor concentration at which 50% inhibition of the enzyme activity occurs) of each encapsulated PHF samples were determined by plotting graph with varying concentrations of the plant extracts against the percent inhibition.

2.9.2. α-Glucosidase Inhibition Assay The α-glucosidase was assayed using a method modified by Apostolidis et al. [31]. Aliquot of 0–4 mg/mL in DMSO (v/v 1:1) of each encapsulated PHF samples were prepared. 50 µL of each concentration sample was mixed well with 100 µL of 0.1 M phosphate buffer (pH 6.9) containing α-glucosidase solution (1.0 U/mL) and the mixtures were then incubated in 96-well plates at 25 ◦C for 10 min. After pre-incubation, 50 µL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25 ◦C for 5 min. Before and after incubation absorbance readings were recorded at 405 nm using a micro-plate reader (Thermomax, Molecular Device Co.) and compared to a control which contained 50 µL of the buffer solution instead of the extracts. The experiments were performed in triplicate and the α-glucosidase inhibitory activity was expressed as percentage inhibition. Acarbose was prepared in distilled water and used as positive controls. The percentage inhibition was calculated using the formula; % Inhibition = {(Ac − Ae)/Ac} 100 where Ac and Ae are the absorbance of the control and extract respectively. IC50 values (inhibitor concentration at which 50% inhibition of the enzyme activity occurs) of each encapsulated PHF samples were determined by plotting graph with varying concentrations of the plant extracts against the percent inhibition.

2.9.3. Determination of Bioavailability of Microencapsulated Products by In Vitro Dialyzability Assay The estimation for bioavailability of all microencapsulated products was determined by the method developed by Pineiro et al. [32].

2.10. Acute Toxicity The acute oral toxicity study was carried out in compliance with Organization for Economic Cooperation and Development (OECD) guideline 425 [33]. All mice (n = 5) for testing were fasted for 12 h and weigh have been recorded and subsequently received the solution of microencapsulated products of PHF at the final concentration of 2000 mg/kg by gavage. The animals were observed individually at least once during the first 30 min after dosing, periodically for first 24 h and regularly thereafter for 14-day of feeding period for gross behavioral changes, toxicity symptoms or mortality. Nutrients 2018, 10, 843 7 of 25

2.11. LC-ESI-QTOF-MS Analyses For LC-ESI-QTOF-MS analysis, firstly ethanolic extract was prepared using PLEx as described in Section 2.4. Afterwards obtained ethanolic extract was used to for the metabolite profiling of PHF using an Agilent 1100 Liquid Chromatography system (Agilent Technologies, Palo Alto, CA, USA) furnished with a standard auto-sampler. The analytical column used was characterized as Phenomenex Gemini C18 (3 µm, 2 × 150 mm) operated at 25 ◦C with a gradient elution portfolio at a flow rate of 0.2 mL/min. The mobile phases used were of acidified water (0.5% acetic acid) (A) and acetonitrile (B). The following multi-step linear gradient applied in following fashion: 0 min, 5% B; 5 min, 15% B; 25 min, 30% B; 35 min, 95% B; 40 min, 5% B. The initial conditions were maintained for 5 min. The injection volume of sample in system was 1µL. The LC-MS system was further composed of a Dionex Ultimate 3000 Rapid Separation LC system coupled to a micrOTOF QII mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an electro-spray source operating in positive mode. The LC system contained an SRD-3400 solvent rack/degasser, an HPR-3400RS binary pump, a WPS-3000RS thermostated auto-sampler, and a TCC-3000RS thermostated column ◦ compartment. The micrOTOF QII source parameters were as follows: temperature, 200 C; drying N2 flow, 8 L/min; nebulizer N2, 4.0 bar; end plate offset, −500 V; capillary voltage, −4000 V; mass range, 50−1500 Da, acquired at 2 scans/s. Post acquisition internal mass calibration used sodium formate clusters with the sodium formate delivered by a syringe pump at the start of each chromatographic analysis. Nitrogen was used as drying, nebulizing and collision gas. The precise mass data of the molecular ions were processed using Data Analysis 4.0 software (Bruker Daltoniks, Bremen, Germany), which delivered a list of potential elemental formulas via the Generate Molecular Formula Editor. The generate molecular formula Editor uses a CHNO algorithm, which deals with standard practicalities such as electron configuration, minimum/maximum elemental range and ring-plus double-bond equivalents, as well as a sophisticated comparison of the theoretical with the measured isotope pattern (Sigma Value, Bruker Daltonics, Bremen, Germany) for increased confidence in the recommended molecular formula. The commonly acknowledged accuracy threshold for validation of elemental compositions was established at 5 ppm [34]. It is significant to point out that even with very high mass precision (<1 ppm) many chemically likely formulas may be found, subjected to the mass regions considered and so high mass accuracy alone is not enough to discount enough candidates with complex elemental compositions. The use of isotopic abundance patterns as a single further constraint, however, eliminates >95% of the false candidates. This orthogonal filter can diminish numerous thousand nominees down to only a small number of molecular formulas. During the development of the HPLC method, the instrument was calibrated externally with a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Chicago, IL, USA) directly linked to the interface and injected with a sodium acetate cluster solution containing 5 mM sodium hydroxide and 0.2% acetic acid in water: isopropanol (1:1, v/v). The calibration solution was injected at the beginning of each run and all the spectra were calibrated prior to compound identification. By using this method, an exact calibration curve based on several cluster masses, each differing by 82 Da (NaC2H3O2) was obtained. Due to the compensation of temperature drift in the micrOTOF-Q II, this external calibration provided accurate mass values of better than 5 ppm for a complete run without the need for a dual sprayer setup for internal mass calibration.

3. Statistical Analysis All statistical analyses were conducted using a one-way analysis of variance using Dunnett’s comparison tests or unpaired t-tests. All calculations were carried out using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Significance was observed at p < 0.05. Nutrients 2018, 10, 843 8 of 25

4. Results and Discussion

4.1. The Effect of Microencapsulation on the Contents of Antioxidant Components and Antioxidant Activity of PHF The contents of antioxidant components of PHF extract treated with different encapsulating wall materials were shown in Table1. In comparison to untreated extract, all microencapsulated treatments have less antioxidant components (i.e., TPC, TFC & TCT). The retention for all freeze-dried treatments demonstrated in the term of percentages, ranged from 94.28% to 68.22% for TPC, 76.46% to 40.35% for TFC and 79.24% to 59.70% for TCT representing the effectiveness of microencapsulation procedure. The wide-ranging powders produced from the microencapsulation process, especially those obtained from TC, retained higher contents of antioxidant components. In general, these results may be associated with the type and concentrations of different wall materials. There were many multifaceted factors which were responsible for hammering of polyphenol compounds during freeze drying method, the crushing of lyophilized microencapsulated products after freeze-drying, were considered one of the key factors which may cause the degradation of bioactive components in the final products by boosting the product’s contact with environment. Our finding was in agreement with previous work in which authors explored that lyophilized wine product contained almost 70% of the original phenolics components [35,36]. Other factors which may responsible for declining the concentration of active components include: formation of microspheres during the lyophilization due to a scattering of the bioactive components inside the configuration of encapsulating wall materials i.e., consisting of one or more constant phase of encapsulating agents [19], development of micro-pores in the aforesaid microspheres, mainly associated to sublimation process during lyophilization [37]. In the current study, lyophilized product encompassed a reduction of 5.72–31.78% for TPC, declined trend of 23.54–59.65% and 20.76–40.30% was also observed for TFC and TCT respectively. Despite the reduction of antioxidant components of microencapsulated products, a significant retentions were also observed (described above in detail with percentages) comparable/higher to prior studies i.e., authors found, that acai pulp microencapsulated with GA have phenolic retention of 94.1% [16]. The freeze dried product microencapsulated with 10% GA (Tc) demonstrated the exceptional conservation for antioxidant components (i.e., TPC, TFC & TCT). The order of effectiveness of microencapsulation for other remaining treatments was as followed: TB > TA > TD. The higher competence of TC treatment was mainly attributed to the structure of GA, because it is a hetero-polymer made up of dense branches of sugar, containing a minute quantity of protein which connected to the carbohydrate skeleton via covalent bonds, proceeding as a tremendous microencapsulating material [38]. Noteworthy results were also found for TB and TA, which might be credited to presence of 5% GA. In contrary, no significant difference was noticed for the lyophilized product having10% MD as wall material (TD). Nutrients 2018, 10, 843 9 of 25

Table 1. Antioxidant components and antioxidant activities of PHF extracts microencapsulated with gum arabic (GA), gelatin (GE), maltodextrin (MD) and their combinations by Freeze-drying method.

Treatments TPC 1 TFC 2 TCT 3 DPPH 4 Beta-carotene 5 ABTS 6 Control 26.72 ± 0.61 a 6.848 ± 0.05 a 15.72 ± 0.3 a 133.3 ± 1.79 a 83.39 ± 0.79 a 3.687 ± 0.03 a b e d d,e d,e b TA 22.89 ± 0.41 2.760 ± 0.03 9.383 ± 0.16 74.73 ± 4.6 64.71 ± 0.64 3.197 ± 0.95 a c c b b c TB 24.26 ± 0.085 4.183 ± 0.07 10.10 ± 0.13 85.0 ± 0.5 78.34 ± 0.51 2.777 ± 0.125 a b b c c c TC 25.26 ± 0.22 5.233 ± 0.15 12.46 ± 0.021 78.11 ± 1.67 75.40 ± 0.88 2.733 ± 0.06 c d d f d,e d,e TD 18.27 ± 0.15 3.817 ± 0.03 9.383 ± 0.07 51.52 ± 0.72 65.72 ± 0.92 2.285 ± 0.072

Note: Results displayed are a representation of triplicate quantifications per extract. TA: Freeze-dried, with 5% GA and 5% GE; TB: Freeze-dried, with 5% GA and 5% MD; TC: Freeze-dried, 1 2 with 10% GA; TD: Freeze-dried, with 10%. Total phenolic contents (TPC) expressed as mg gallic acid equivalents (GAE) per g of dry extract; Flavonoid content expressed as mg quercetin equivalents (QE) per g of dry extract; 3 Total condensed tannin content based on calibration curve of (+)-catechin, expressed as mg catechin equivalents (CE) per g of dry 4 5 6 extract. DPPH expressed as µmol/g sample on dry basis; β-carotene of extracts (5 mg/mL) based on percent bleaching inhibition. EC50 (mg/mL) is representative of the effective concentration at which 50% of ABTS+ radicals were scavenged. The Dunnett’s test was to evaluate the significance with confidence level was set to 95%; different letters within the same column indicate significant differences (p < 0.05). Nutrients 2018, 10, 843 10 of 25

The antioxidant activity for microencapsulated powders determined by DPPH, β-carotene and ABTS+ assay were illustrated in Table1. All microencapsulated products had showed decrease antioxidant by DPPH assay in relation to original extract (control) and their retention ranged from 38.84–64.50%. TB (5% GA & 5% MD) and Tc (10% GA) illustrated the highest antioxidant activity; these results were agreement with previously found values by Souza et al. [39]. The order for effectiveness was noticed as: TB > TC > TA > TD. In the case of β-carotene bleaching assay, the antioxidant retention for all microencapsulated products were explored from 77.59% to 93.93% in comparison to original extract, TB (5% GA & 5% MD) showed maximum value for antioxidant activity in a similar way as in DPPH assay. Remaining treatments have been categorized in context of efficacy as followed: + TC > TD > TA. Referring to antioxidant assay by ABTS radical scavenging activity, the range of retention was from 62.2% to 86.68%. The noteworthy consequence was revealed for TA (5% GA & 5% GE), while TB (5% GA & 5% MD) and TC (10% GA) also illustrated the significant results with retention of 75.27% and 74.18% respectively. The above discussion suggested the worthiness of diverse antioxidant assay for secure and overwhelming conclusion, because each assay comprised its own preciseness and proceeds at a challenging site of action. Amongst the all lyophilized encapsulated products, the antioxidant activity was higher in TB and TC, being related to the presence of high antioxidant components (i.e., TPC, TFC & TCT) (Table1), which provided an excellent defense system against unrestrained oxidation, owing to its high reducing power. Furthermore, there is no report yet on microencapsulation of aforesaid polyphenol enriched extract from PHF and their characterization related to analysis for antioxidant.

4.2. Physical Characteristics of Microencapsulated Powder Products Physical factors i.e., water activity; moisture contents and hygroscopicity are indispensable for encapsulating products steadiness and storage, whilst aqueous solubility is correlated with ability of powder products for reconstitution [18]. The moisture contents for four different lyophilized encapsulated products were demonstrated in Figure1A. The moisture content of said powders were ranged from 7.07% to 9.04%; on the contrary, no significant difference was found between TB and TD (7.41% and 7.21%, respectively). Our findings was validated by earlier investigation which elaborated the moisture contents for blackberry fruit drink encapsulated by means of MD and trehalose dehydrate were of 2.44–6.11% [40]. Lower freezing temperature i.e., less than −40 ◦C consequences in quick freezing, eventually caused tiny pores in the superficial coatings, which might encumber the mass transfer and regarded as an obstacle for sublimation process, causing the higher retention of moisture contents in microencapsulated products [41]. The water activity (aw) of all microencapsulated products (Figure1B) was ranged from 0.310 to 0.450, and all final encapsulated products were noticeably dissimilar from one another, apart from TB (5% GA & 5% MD). TD (10% MD) demonstrated the maximum aw value of 0.450 which was corroborating with previous study carried out by Gurak et al. [42] who found that aw of grape fruit drink microencapsulated by the means of maltodextrin utilizing lyophilization technique was 0.430. Various factors that determine the solubility of the microencapsulated powdered products includes: the feed composition and particle size. The selection of the wall material is very important, not only for the solubility itself but also to the crystalline state that ultimately bestowed to the dried powders [43]. The aqueous solubility for all lyophilized treatments was ranged from 84.06% to 92.31% as illustrated in Figure1C. The solubility of the final product possibly not only associated with solubility prospective of microencapsulating wall material but also on attainted particle size in final desirable product; if particle size would be minute, it would ultimately provide the better surface area’s availability for the hydration process [44,45]. The highest solubility value was obtained for treatment TD (10% MD) that was consistent with previous work. Moreira et al. [20] elaborated the solubility percentage for acerola pomace extract ranged from 90.97 to 96.92%, using MD and tree’s gum of cashew apple as microencapsulating wall materials. Nutrients 2018, 10, 843 11 of 25 Nutrients 2018, 10, x FOR PEER REVIEW 12 of 24

A D 10 20 *** ** 8 * ** * 15 *

6 O/100g dry solids) 2 10

4 Hygroscopicity (%) 5 2 Moisture Contents(g H 0 0 l l o A B C D o A B C D tr T T T T tr T T T T n n o o C C

B E 0.6 50 ***

*** C)

o 40 ** )

w 0.4 ** ** 30 **

* 20 0.2 Water Activity (A

10 Glass transition temperature (

0.0 0

l l o A B C D o A B C D tr T T T T tr T T T T n n o o C C

C F 80 100 * * * 80 60 * *

60 40

40 Solubility (%)

Bioavailablity ratio (%) 20 20

0 0 l l o T A B T C T D o A B C D tr T tr T T T T n n o o C C Figure 1. (A–F) Physical properties and bioavailability ratio (%) of PHF extract microencapsulated Figure 1. (A–F) Physical properties and bioavailability ratio (%) of PHF extract microencapsulated with GA, GE, MD and their combinations by freeze-drying method. Treatment A (TA): Freeze-dried, with GA, GE, MD and their combinations by freeze-drying method. Treatment A (TA): Freeze-dried, with 5% GA & 5% GE; Treatment B (TB): Freeze-dried, with 5% GA & 5% MD; Treatment C (TC): with 5% GA & 5% GE; Treatment B (TB): Freeze-dried, with 5% GA & 5% MD; Treatment C (TC): Freeze-dried, with 10% GA; Treatment D (TD): Freeze-dried, with 10% MD. Freeze-dried, with 10% GA; Treatment D (TD): Freeze-dried, with 10% MD.

Nutrients 2018, 10, 843 12 of 25

The hygroscopicity values for all microencapsulated powder products by the means of freeze-dying method were depicted in Figure1D. These were ranging from 11.92% to 14.35%, representing a lesser amount of hygroscopicity values for powder products; hence assisted the protection of antioxidant components. The findings of current work have much resemblance with preceding work, utilizing related sort of microencapsulating wall materials. Some renowned investigators reported the hygroscopicity of microencapsulated products made up from bark extract of jaboticaba tree using MD and GA as wall material of 17.75%. The lyophilized powdered products demonstrated the lesser hygroscopic values, regardless the presence of higher contents of moisture [15]. The aforesaid behavior was also reported by Khazaei, et al. [46]. The lower values of hygroscopicity for the all lyophilized products mainly attributed to the bigger particle size, since the bigger the particle size, the lesser the uncovered surface area, therefore low down the water absorption [16,47]. The stability of microencapsulated powdered products for the period of storage was principally determined by glass transition temperatures (Tg), the lower the Tg resulting in lower the stability of final product and vice versa. The glass transition temperatures of all lyophilized products were of 15.86 to ◦ 45.0 C in range (Figure1E). Amongst all lyophilized microencapsulated products, the T C represented the highest glass transition temperature (45.0 ◦C), proving maximum stability. Furthermore, other treatments also showed significant values for Tg except TD. The glass transition temperature has been influenced by diverse factors, including moisture contents, chemical configuration and molecular mass of subjected matter [48]. Adhikari et al., 2004 found the lower transition temperatures of fruit drinks/extract were mainly due to the existence of elevated quantity of low molecular weight organic acids and polysaccharides [49]. Additionally, integration of microencapsulating agents in extracts has much predisposed on glass transition temperatures which varied according to molecular weight of encapsulating material; increase in molecular weight of wall material resulting the increase in final Tg of the product. The results of our current work were corroborated with earlier findings [50–52]. The lyophilized microencapsulated product obtained from treatment D (TD) represented the lower Tg because of lower molecular weight of MD. Moreover, this behavior was not noticed in TC (10% GA), TA (10% GA & 5% GE) and TB (10% GA & 5% MD) due to the existence of uppermost molecular weight of GA in the term of quality and quality of wall material.

4.3. Bioavailable TP Contents TP bioavailability ratios, articulated in the term of percentage, were computed by using the equation as followed: [TP]Dialyzable Bav(%) = × 100 [TP] Total where, Bav (%) represented the percentage (%) for TP bioavailability, whereas [TP] Total and [TP] Dialyzable demonstrated TP concentrations after the PLE extraction method and in vitro digestion procedure respectively. Figure1F depicted the bioavailability ratio (%) for all freeze-dried microencapsulated products. Treatment TA and TC demonstrated the excellent bioavailability ratios (%) i.e., 57.25 and 54.64% respectively; there was no significant difference in TB and TD. Furthermore, no research has yet been conducted on in vitro dialyzability analysis of aforesaid microencapsulated PHF products.

4.4. α-Amylase & α-Glucosidase Inhibition Type-II DM an outcome of insulin resistance is a metabolic disease that, according to the latest data for the World Health Organization in 2014, impinges on 9% of the world’s population, both in developed and developing countries, and directly caused 1.5 million deaths in that single year [53,54]. In order to hamper the side effects of type-II DM, insulin injection and usage of anti-hyperglycaemic substances are two key conventional approaches. The management of the blood sugar level is effective and novel approach to overcome the diabetes mellitus and related complications. Inhibitors of carbohydrate hydrolyzing enzymes (i.e., α-amylase and α-glycosidase) have been practically valuable Nutrients 2018, 10, 843 13 of 25 as oral hypoglycemic drugs and regarded as a reliable indicator for the efficacy of therapeutic agents [55,56]. Several α-amylase inhibitors including acarbose, miglitol and voglibose are clinically useful to treat diabetes but these are expensive and have considerable clinical side effects. Medicinal plants have great potential to retard the absorption of glucose by inhibiting the saccharides hydrolyzing enzymes [57–60]. There was an attempt to explore the remarkable drugs from medicinal plants featured with elevated potency and less adverse effects than existing drugs [61,62]. Therefore, screening and isolation of inhibitors from plants for these enzymes are escalating. In the aforementioned context, our microencapsulated polyphenolic enriched powders were investigated for α-amylase and α-glycosidase inhibition as shown in Figure2A,B. Diverse classes of polyphenolic compounds in the current PHF extract were detected likewise: flavonoids, alkaloids, terpenoids, lignans, glycerophospholipid, prenol lipids and their derivatives (detailed in Section 4.7), which eventually may be considered for anti-diabetic potential of microencapsulated powders of current study. The treatment TC (10% GA) demonstrated the highest inhibition at concentration of 4 mg/mL, for α-amylase (93.33 ± 2.65, with IC50 value 1.47 mg/mL ± 0.57) and α-glucosidase (73.39 ± 1.66 with IC50 value 2.03 ± 0.45 mg/mL), representing highest anti-diabetic potential. Previously, none of investigation has yet been carried out on lyophilized aforementioned microencapsulated PHF products. Additionally, there is no report on microencapsulation of polyphenol enriched extract from PHF and their characterization for anti-diabetic potential purposes, which eventually facilitate to take decision for commercialization of microencapsulated products i.e., polyphenolsNutrients 2018, 10 enriched, x FOR PEER nutrient REVIEW supplement. 13 of 24

A 100 ** *** ** * *** * 80 *** *** *** ** * *** *** 60 *** **

40 ***

Control

TA 20 TB Inhibitionrateon alpha-amylase (%) TC TD

0 0 1 2 3 4 Concentration (mg/mL)

B Figure 2. Cont. 80 * * ** * * *** ** 60 *** ***

*** **

40 ***

20 Inhibitionrate on alpha-glucosidase (%)

0 0 1 2 3 4 Concentration (mg/mL) Figure 2. (A,B) α-amylase and α-glucosidase inhibition activities of PHF extract microencapsulated with GA, GE, MD and their combinations by freeze-drying method. Control: Acarbose; Treatment A (TA): Freeze-dried, with 5% GA & 5% GE; Treatment B (TB): Freeze-dried, with 5% GA & 5% MD; Treatment C (TC): Freeze-dried, with 10% GA; Treatment D (TD): Freeze-dried, with 10% MD.

4.5. Size Distribution and Morphology of Microencapsulated Powders Different polymers exhibited particular protection capacity, so the evaluation of microencapsulated products is very crucial. This aforesaid capacity elaborated the extent of micro- pores and reliability of encapsulated micro-particles [63]. The structural analysis of the encapsulated products from the lyophilization methodology was conducted by the means of scanning electron microscope (Quanta 250 EFI). Comparison of the images illustrated the noticeable variation in term of particle structure and size allocation amongst the different microencapsulated products and their

Nutrients 2018, 10, x FOR PEER REVIEW 13 of 24

A 100 ** *** ** * *** * 80 *** *** *** ** * *** *** 60 *** **

40 ***

Control

TA 20 TB Inhibitionrateon alpha-amylase (%) TC TD

0 Nutrients 2018, 10, 843 0 1 2 3 4 14 of 25 Concentration (mg/mL) B 80 * * ** * * *** ** 60 *** ***

*** **

40 ***

20 Inhibitionrate on alpha-glucosidase (%)

0 0 1 2 3 4 Concentration (mg/mL)

Figure 2. (A,B) αα-amylase-amylase and αα-glucosidase-glucosidase inhibition activities of PHF extractextract microencapsulatedmicroencapsulated with GA, GE, MD MD and and their their combinations combinations by by freeze-drying freeze-drying method. method. Control: Control: Acarbose; Acarbose; Treatment Treatment A A B A(T (T):A Freeze-dried,): Freeze-dried, with with 5% 5% GA GA & &5% 5% GE; GE; Treatment Treatment B B (T (T):B ):Freeze-dried, Freeze-dried, with with 5% 5% GA GA & & 5% 5% MD; MD; C D Treatment CC (T(TC): Freeze-dried, Freeze-dried, with 10% GA; Treatment DD (T(TD):): Freeze-dried, Freeze-dried, with with 10% 10% MD.

4.5. Size Distribution and Morphology of Microencapsulated Powders Different polymers polymers exhibited exhibited particular particular protection protection capacity, so capacity, the evaluation so of the microencapsulated evaluation of productsmicroencapsulated is very crucial. products This is aforesaid very crucial. capacity This elaborated aforesaid capacity the extent elaborated of micro-pores the extent and reliabilityof micro- poresof encapsulated and reliability micro-particles of encapsulated [63]. micro-particlesThe structural analysis[63]. The ofstructural the encapsulated analysis of products the encapsulated from the productslyophilization from methodologythe lyophilization was conductedmethodology by thewas means conducted of scanning by the electronmeans of microscope scanning (Quantaelectron microscope250 EFI). Comparison (Quanta 250 of EFI). the images Comparison illustrated of the the images noticeable illustrated variation the noticeable in term of particlevariation structure in term ofand particle size allocation structure amongst and size the allocation different amongst microencapsulated the different products microencapsulated and their combination products and attained their after lyophilization. Figure3A–D demonstrated the morphology of all freeze-dried microencapsulated products. As can be seen all lyophilized products presented the irregular shape like broken glass with appreciable proportion of pores on surface. The outcome of current investigation has agreement with the recent work explored by Kuch and Norena [64]. These authors studied on morphological aspects of lyophilized products, made up from the peel of grapes and pomace of Averrhoa carambola and presented the final product as porous, uneven and brittle conformation; furthermore they also described the reason behind the high porosity of lyophilized products as development of ice crystals had happen in material which as a result retarded the breakdown of final configuration and hence less change in volume occurred. There was a direct association between span value and dispersal of particle size, the lesser span value demonstrating a uniform distribution of micro-particles [65]. The size of micro-particles from the final products was in the range of 18.08 to 391.30 µm. TA explored the higher particle size (more than 287 µm), whereas TD showed the lowest one (Table2). Our current work is consistent with prior investigation, examined by other authors [57] who found that the particle size of microencapsulated product via freeze-dying method reached up to 300 µm. The bigger particle dimension of lyophilized products was mainly attributed to rapid freezing and less availability of force to crush the freeze drop during lyophilization [66,67]. Moreover, particle size was also influenced by crushing procedure which was generally accustomed for size reduction after lyophilization. Nutrients 2018, 10, x FOR PEER REVIEW 14 of 24 combination attained after lyophilization. Figure 3A–D demonstrated the morphology of all freeze- dried microencapsulated products. As can be seen all lyophilized products presented the irregular shape like broken glass with appreciable proportion of pores on surface. The outcome of current investigation has agreement with the recent work explored by Kuch and Norena [64]. These authors studied on morphological aspects of lyophilized products, made up from the peel of grapes and pomace of Averrhoa carambola and presented the final product as porous, uneven and brittle conformation; furthermore they also described the reason behind the high porosity of lyophilized productsNutrients 2018 as, 10 development, 843 of ice crystals had happen in material which as a result retarded15 of the 25 breakdown of final configuration and hence less change in volume occurred.

(A) Treatment-TA (B) Treatment-TB

(C) Treatment-TC (D) Treatment-TD

Figure 3. (A(A––DD) ) Micrographs Micrographs of of PHF PHF extract extract microencapsulated microencapsulated with with GA, GA, GE, GE, MD MD and and their combinations by freeze-drying method. (A) Treatment-TA: Freeze-dried, with 5% GA & 5% GE; (B) combinations by freeze-drying method. (A) Treatment-TA: Freeze-dried, with 5% GA & 5% GE; Treatment-TB: Freeze-dried, with 5% GA & 5% MD; (C) Treatment-TC: Freeze-dried, with 10% GA; (B) Treatment-TB: Freeze-dried, with 5% GA & 5% MD; (C) Treatment-TC: Freeze-dried, with 10% GA; (D) Treatment-TD: Freeze-dried, with 10% MD. (D) Treatment-TD: Freeze-dried, with 10% MD.

There was a direct association between span value and dispersal of particle size, the lesser span valueTable demonstrating 2. Average diameter a uniform and distribution particle size distributionof micro-particles (Span) of [65]. the PHFThe extractsize of microencapsulated micro-particles from the finalwith products GA, GE, MD was and in theirthe range combinations of 18.08 by to freeze-drying 391.30 μm. method.TA explored the higher particle size (more than 287 μm), whereas TD showed the lowest one (Table 2). Our current work is consistent with prior investigation, examinedTreatments by other authors Average [57] who Diameter found (µ m)that the particle Span size of microencapsulated product via freeze-dying methodTA reached up to151.13 300 μm. The bigger particle 1.74 dimension of lyophilized products was mainly attributedTB to rapid freezing76.15 and less availability of 1.21force to crush the freeze drop T 92.79 2.88 during lyophilization [66,67].C Moreover, particle size was also influenced by crushing procedure TD 18.95 1.52 which was generally accustomed for size reduction after lyophilization. Treatment A (TA): Freeze-dried, with 5% GA and 5% GE; Treatment B (TB): Freeze-dried, with 5% GA and 5% MD; Treatment C (TC): Freeze-dried, with 10% GA; Treatment D (TD): Freeze-dried, with 10% MD.

4.6. Acute Toxicity No toxic effects and mortality were observed at a dose of 2000 mg/kg by gavage. Consequently, microencapsulated products of PHF extract were regarded as safe for consumption.

4.7. Bioactive Compounds from LC-ESI-QTOF-MS Analysis The ethanolic extract of freeze dried fine powder of PHF was a multifaceted mixture of compounds. Figure4 characterized the chromatogram of said ethanolic extract. The bioactive compounds were recognized by means of the comparing retention times (RT) and MS/MS spectra granted by QTOF-MS with those of valid standards wherever obtainable and via elucidation of MS and MS/MS spectra from QTOF-MS merged with data available in literature. MS data of identified compounds has been recapitulated in the Table3 including calculated m/z for molecular formulas provided, main fragment Nutrients 2018, 10, x FOR PEER REVIEW 15 of 24

Table 2. Average diameter and particle size distribution (Span) of the PHF extract microencapsulated with GA, GE, MD and their combinations by freeze-drying method.

Treatments Average Diameter (µm) Span TA 151.13 1.74 TB 76.15 1.21 TC 92.79 2.88 TD 18.95 1.52

Treatment A (TA): Freeze-dried, with 5% GA and 5% GE; Treatment B (TB): Freeze-dried, with 5% GA and 5% MD; Treatment C (TC): Freeze-dried, with 10% GA; Treatment D (TD): Freeze-dried, with 10% MD.

4.6. Acute Toxicity No toxic effects and mortality were observed at a dose of 2000 mg/kg by gavage. Consequently, microencapsulated products of PHF extract were regarded as safe for consumption.

4.7. Bioactive Compounds from LC-ESI-QTOF-MS Analysis The ethanolic extract of freeze dried fine powder of PHF was a multifaceted mixture of compounds. Figure 4 characterized the chromatogram of said ethanolic extract. The bioactive compounds were recognized by means of the comparing retention times (RT) and MS/MS spectra grantedNutrients 2018by ,QTOF-MS10, 843 with those of valid standards wherever obtainable and via elucidation of16 ofMS 25 and MS/MS spectra from QTOF-MS merged with data available in literature. MS data of identified compounds has been recapitulated in the Table 3 including calculated m/z for molecular formulas provided,obtained by main MS/MS, fragment error obtained and proposed by MS/MS, compound error and for proposed each peak. compound Diverse classes for each of peak. polyphenolic Diverse classescompounds of polyphenolic have been discoveredcompounds in have the ethanolic been discovered extract of in PHF. the ethanolic Annotated extract compounds of PHF. represented Annotated compoundsthe diverse classes represented includes the flavonoids, diverse classes alkaloids, includes terpenoids, flavonoids, lignans, alkaloids, glycerophospholipid terpenoids, lignans, and glycerophospholipidprenol lipids. and prenol lipids.

FigureFigure 4. 4. ChromatogramChromatogram of of the the Ethanolic Ethanolic extract extract derived derived from from freeze freeze dried powder of PHF.

Peaks 4, 8, 9, 10, 11, 12, 14, 15, 17, 18, 19, 28, 32, 36, 39, 41, 44 and 45 represented different flavonoid compounds and their derivatives which possess diverse previously proven biological activities i.e., anti-inflammatory, anti-nociceptive, anti-oxidative, anti-dengue, anti-malarial [68–74]. Among them 3 bioactive compounds (peak 9, 18 and 39) were classified as 6-prenylated flavones (i.e., flavones that features a C5-isoprenoid substituent at the 6-position). These bioactive compounds are insoluble in aqueous solution and designated as a faintly acidic compound. These compounds previously found in fruits, peas and pulses and considered to be flavonoid lipid molecules. While some compounds (peak 17, 41, 44 and 45) belong to sub class flavonoids glycosides likewise; quercetagetin 7-glucoside (compound 17, m/z 481.2572 [M + H]) and quercetin 3-(600-malonylglucoside)-7-glucoside (Compound 41, m/z 713.5121 [M + H]) were recognized as flavonoid-7-o-glycosides. These are phenolic compounds containing a flavonoid moiety which is O-glycosidically linked to carbohydrate moiety at the C 7-position. These derivatives of flavonoids have priory proved strong antioxidative, anticancer, neuro-protective, anti-inflammatory, diuretic, hypoglycemic and anti-hepatitis activities [75,76]. Moreover, catalpol m/z 363.195 [M + H] (compound 15) demonstrated a variety of biological activities including anti-cancer, neuro-protective, anti-inflammatory, diuretic, hypoglycemic and anti-hepatitis virus effects. Previous studies have also provided some clues that catalpol can affect energy metabolism through increasing mitochondrial biogenesis, enhancing endogenous antioxidant enzymatic activities and inhibiting free radical generation ultimately attenuates oxidative stress [77]. Nutrients 2018, 10, 843 17 of 25

Table 3. Bioactive Compounds identified in Ethanolic Extract of PHF.

Peak No. RT (min) Assigned Compound Name Elemental Composition m/z [M + H]+ Difference (mDa)

1 8.773 Caffeic acid 4-sulfate C9H8O7S 261.128 0.71 2 9.458 Steviol C20H30O3 319.1329 −0.92 3 9.708 Antherospermidine C18H11NO4 305.1541 0.15 4 9.904 Eriodictyol C15H12O6 289.1231 −0.14 5 9.95 Phloretin C15H14O5 275.1077 1.04 6 10.177 Zanthobisquinolone C21H18N2O4 363.1589 0.57 7 10.384 Murrayazolinol C23H25NO2 349.1795 0.72 8 10.639 Patuletin C16H12O8 333.1488 0.34 9 10.746 Albanin d C25H26O5 407.1849 0.84 0 0 0 10 10.834 3,5,8,3 ,4 ,5 -Hexahydroxyflavone C15H10O8 319.1329 0.31 11 10.918 Myricetin C15H10O8 319.1692 0.3 12 10.936 Dehydroneotenone C19H12O6 393.2055 0.77 13 11.219 Carissanol C20H24O7 377.1746 0.86 14 11.4 Epigallocatechin 3-O-cinnamate C24H20O8 437.2313 −0.13 15 11.479 Catalpol C15H22O10 363.195 0.8 16 11.528 Secoisolariciresinol C20H26O6 363.1589 0.46 17 11.805 Quercetagetin 7-glucoside C21H20O13 481.2572 −0.02 18 11.945 Cajaflavanone C25H26O5 407.2208 −0.17 19 12.163 Barbatoflavan C24H28O13 525.2828 0.9 20 12.348 Celastrol C29H38O4 451.2469 0.04 21 12.484 6-Gingerol C17H26O4 296.1487 −1.92 22 12.77 Euphorbia diterpenoid 3 C33H40O11 613.3348 0.89 23 13.017 2-Hexaprenyl-6-methoxyphenol C37H56O2 534.3435 0.67 24 13.278 PE(P-16:0/18:2(9Z,12Z)) C39H74NO7P 701.3867 −1.1 25 13.569 Buddliedin A C17H24O3 277.1385 −0.24 26 16.104 Phytosphingosine C18H39NO3 318.2974 −0.04 27 17.059 1-Eicosenee C20H40 282.2015 0.84 28 19.53 Tectorigenin C16H12O6 301.1391 0.18 29 20.643 3-O-cis-Coumaroylmaslinic acid C39H54O6 619.3973 −0.4 30 20.884 PA(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)) C41H69O8P 721.4644 0.32 Nutrients 2018, 10, 843 18 of 25

Table 3. Cont.

Peak No. RT (min) Assigned Compound Name Elemental Composition m/z [M + H]+ Difference (mDa)

31 21.141 Eurysterol A sulfonic acid C27H46O7S 515.3518 0.33 32 21.548 Citflavanone C20H18O5 338.3391 1.25 33 21.698 Mesaconitine C33H45NO11 631.4345 −0.17 34 22.01 Xanthoangelol C25H28O4 393.294 0.44 35 22.385 PA(15:0/22:4(7Z,10Z,13Z,16Z)) C40H71O8P 711.4757 0.56 0 36 22.589 2 ,5,6-trimethoxyflavone C18H16O5 312.3236 1.42 37 22.613 Ubiquinol-8 C49H76O4 729.5073 0.38 38 22.646 Epicalyxin J C42H38O9 686.4852 0.14 39 22.967 Luteone C20H18O6 354.37 1.64 0 40 23.177 Luteolin 4 -sulfate C15H10O9S 366.3702 1.43 00 41 23.524 Quercetin 3-(6 -malonylglucoside)-7-glucoside C30H32O20 713.5121 0.64 42 24.348 Phytoene C40H64 545.1143 0.39 0 43 26.083 Epigallocatechin 3,3 ,-di-O-gallate C29H22O15 610.1796 0.62 00 00 00 44 26.819 Kaempferol 3-(2 ,3 -diacetyl-4 -p-coumaroylrhamnoside) C34H30O14 663.4496 0.47 00 45 29.446 Delphinidin 3-(6 -malonyl-glucoside) C24H23O15 684.1982 0.21 Nutrients 2018, 10, 843 19 of 25

Mesaconitine (peak 33, m/z 631.4345 [M + H]) and antherospermidine (peak 3, m/z 305.1541 [M + H]+) were the member of group named alkaloids, later have a structure that contains an aminoethylphenanthrene moiety. Atherosperminine has been cited to be in fruits and bark of Cryptocarya nigra (Lauraceae) and have strong antioxidant, anti-mlarial and anti-microbial activities [67]. Steviol, m/z 31 9.1329 [M + H] designated as (peak 2) in our list of metabolites, is diterpene alkaloids with a structure that is based on the kaurane skeleton. It possesses a [3, 2, 1]-bicyclic ring system with C15-C16 bridge connected to C13, forming the five-member ring D. This compound was excessively found in different sorts of fruits and primarily responsible for the sweet taste of stevia leaves. This compound is considered safe for human consumption and was approved as a food additive by the Food and Drugs Administration (FDA) and European Food Safety Authority (EFSA) and helped to reduce the oxidative stress [78]. In addition, the peak 6 is of zanthobisquinolone, m/z 363.1589 [M + H] and peak 7 is of murrayazolinol, m/z 349.1795 [M + H] belongs to the class quionlines and their derivatives, also alkaloid in nature. These are usually present in herbs, spices and some fruits [79–81]. Various anti-malarial, anti-parasitic, anti-bacterial and anti-viral drugs do contain a major constitute of aforesaid bioactive compound [82]. Besides flavonoid and alkaloids, these are also some compounds which have appreciable share, belong to class prenol lipids (peak 20, 23, 37 and 42). Amongst, ubiquinol 8 (peak 37, m/z 729.5073 [M + H]+ belongs to organic compounds known as polyprenyl quinols. It is the reduced configuration of ubiquinone-8. It plays a function as an electron transporter in mitochondrial membrane, where it carries two electrons from either complex I (i.e., NADH dehydrogenase) or complex II (i.e., succinate-ubiquinone reductase) to complex III, whilst, compound 42, named phytoene fragmented at m/z 545.1143 [M + H] is member of class regarded as carotenes and further belongs to family carotenoids. These are unsaturated hydrocarbons comprising of eight repeated isoprene units. They have also previously proven antioxidant, anti-cancer activity and facilitate to reduce the complications [83]. Amongst the known natural bioactive compounds, terpenoids are considered to be of approximately 60%. Plant terpenoids are extensively used for their aromatic qualities and play a role in traditional herbal remedies, for instance; Euphorbia diterpenoids 3 (peak 22) possesses a variety of different core frameworks and exhibit a diverse array of beneficial activities, including anti-tumor, anti-inflammation, and immune-modulatory features and regarded as excellent source in term of scientific attraction [84]. On the other hand, Buddledin A (peak 25) a sesquiterpenoid based on a humulane skeleton, displaying selective anti-fungal activity against dermatophytes [85], while 3-O-cis-coumaroylmaslinic acid (peak 29) have ability to attenuate oxidative stress. Lignans were usually found in fruits and have proved strong anti-cancer and antioxidant activities. Among them, compound 16 (secoisolariciresinol) and compound 13 belong to class dibenzylbutane lignans and furanoid lignans respectively. It was present in a number of food items such as American butterfish, Brazil nut, fireweed, and oriental wheat [86,87]. Besides these, peak 5 and 34 represented phloretin and xanthoangelol respectively. These compounds showed various biological activities i.e., anti-tumor and anti-metastatic features [88,89]. Other detected compounds which were not discussed in detailed such as peak 21, 24, 26, 27, 30, 31 35, are intermediate products of either metabolism or biosynthesis of amino acids, (phosphor or/and sphingo) lipids or others. For instance, metabolite 24th represented a phosphatidylethanolamine, is an anchor protein, produced as an intermediate in gycosylphosphatidylinositol (GPI) anchor biosynthesis pathway, while compound 30 and compound 35 are the phosphatidic acids, produced in glycerolipid biosynthesis. The existences of such compounds are mainly attributed to the seeds of Hygrophila spinosa T. Anders [90]. The 26th peak recognized as phytosphingosine, m/z 318.2974 [M + H], is an intermediate compound synthesized between dihrdro-shingosine and phyto-ceramide in shingophospholipid metabolism. Phospholipids have diverse functions in varied processes of cell i.e., apoptosis, cell propagation, cell to cell interaction, differentiation etc. Furthermore, phytosphingosine is naturally occurring sphingoid bases, fungi and plants are the rich source of phytosphingosine. Nutrients 2018, 10, 843 20 of 25

It is structurally similar to sphingosine; phytosphingosine possesses a hydroxyl group at C-4 of the sphingoid long-chain base. Phyto-sphingosine induces apoptotic cell death in human cancer cells by direct activation of caspase 8, and by mitochondrial translocation of Bax and subsequent release of cytochrome C into cytoplasm, providing a potential mechanism for the anti-cancer activity of phytosphingosine. The metabolite 01, m/z 261.128 [M + H] have been referred to caffeic acid 4-sulfate (polyphenol) belongs to a class cinnamic acids and their derivatives. Hydroxycinnamic acids are compounds containing a cinnamic acid where the benzene ring is hydroxylated. It is one of the most representative phenolic acids in fruits and vegetables which have excellent antioxidative potential and anti-carcinogenic activity [90–93]. As can been concluded that the current PHF is the mixture of previously proven [21–24] health promoting herbs’ parts, so diversity and abundance of such detected antioxidant substances/ metabolites not only made sense but also verify the outcomes. Taking together, this is the first study which exploited the metabolite profiling of said PHF enriched with antioxidants and their evaluation for bioavailability and anti-diabetic potential after encapsulation.

5. Conclusions In the current study, PHF polyphenolic extract was microencapsulated by utilizing GA, GE, and MD as encapsulating wall materials, due to which resulting microcapsules found to have withholding capacity of TPC more than 85% except TD (68.22%), while conserving range of TFC and TCT were found near to 60% except TA. Elevated antioxidant activity was also revealed for TB and TC and reasonable for TA and TD, representing noteworthy and positive correlation of antioxidant assays to all aforementioned antioxidant components. Taking all results into consideration, TB (5% GA & 5% MD) and TC (10% GA) showed the best performance attributable with respect to the higher preservation of antioxidant components and antioxidant activity by means of DPPH and β-carotene assays and significant for an ABTS+ radical scavenging activity, augmented by low contents of moisture, water activity (aw), particle dimension and elevated solubility, hygroscopicity and Tg. Additionally, the aforementioned treatments also demonstrated the excellent morphological features with asymmetrical (irregular) micro-particle structures, depicted lower prevalence of coarseness and crankiness. Moreover, TB,TC and TA also characterized the highest anti-diabetic potential by reason of their significant inhibition rate for α-amylase and α-glucosidase. In the context of bioavailability, TB and TC also demonstrated the excellent bioavailability ratios (%) (i.e., more than 50% & 40% respectively). The bioavailability data revealed that microencapsulation of PHF (especially with TC and TB) can improve the bioavailability of pH and thermo-labile bioactive compounds at intestinal level which is a major site for absorption of bioactive compounds. In addition, no mice proved any toxicity sign at a dose of 2000 mg/kg by gavage for any treatment. In the conclusive manner, we recommended the TB and TC as result of their incredible capability for preserving antioxidant components to its usage in nutraceutical and functional products while masking the undesirable flavor distinctiveness of herbs/herbal extracts.

Author Contributions: Y.S. supervised this work. S.A.H. and A.H. performed all the experimental work. S.A.H. wrote this manuscript. H.A.R.S. edited and reviewed the whole manuscript and provided suggestions to main authors about overall research plan. All authors read and approved the final manuscript. Y.N., T.N. and Y.W. assisted in anti-diabetic assay and data analysis. Funding: This work was supported by National Natural Science Foundation of China (Grant Nos. 31670064 and 31271812), and TaiShan Industrial Experts Program. Acknowledgments: We are indebted to herbal medicine practitioner Syed Ijaz Hussain for kindly providing the specimen herbal plants and helping in formulating the PHF. Conflicts of Interest: Authors declare no conflicts of interests. Nutrients 2018, 10, 843 21 of 25

References

1. Abbas, M.; Saeed, F.; Anjum, F.M.; Afzaal, M.; Tufail, T.; Bashir, M.S.; Ishtiaq, A.; Hussain, S.; Suleria, H.A.R. Natural polyphenols: An overview. Int. J. Food Propert. 2016, 20, 1689–1699. [CrossRef] 2. Hameed, A.; Hussain, S.A.; Yang, J.; Ijaz, M.U.; Liu, Q.; Suleria, H.A.R.; Song, Y. Antioxidants Potential of the Filamentous Fungi (Mucor circinelloides). Nutrients 2017, 9, 1101–1121. [CrossRef][PubMed] 3. Suleria, H.A.; Butt, M.S.; Anjum, F.M.; Saeed, F.; Khalid, N. Onion: Nature protection against physiological threats. Crit. Rev. Food Sci. Nutr. 2015, 55, 50–66. [CrossRef][PubMed] 4. Hurrle, S.; Hsu, W.H. The etiology of oxidative stress in insulin resistance Biomed. J. 2017, 40, 257–262. 5. Yousaf, S.; Butt, M.S.; Suleria, H.A.; Iqbal, M.J. The role of green tea extract and powder in mitigating metabolic syndromes with special reference to hyperglycemia and hypercholesterolemia. Food funct. 2014, 5, 545–556. [CrossRef][PubMed] 6. Ameer, K.; Shahbaz, H.M.; Kwon, J.H. Green extraction methods for polyphenols from plant matrices and their byproducts: A review. Compr. Rev. Food Sci. Food Saf. 2017, 16, 295–315. [CrossRef] 7. Mojzer, E.B.; Knez Hrnˇciˇc,M.; Škerget, M.; Knez, Ž.; Bren, U. Polyphenols: Extraction methods, antioxidative action, bioavailability and anticarcinogenic effects. Molecules 2016, 21, 901. [CrossRef][PubMed] 8. Spinella, M. The importance of pharmacological synergy in psychoactive herbal medicines. Altern. Med. Rev. 2002, 7, 130–137. [PubMed] 9. Chorgade, M.S. Drug Discovery and Development; John Wiley and Sons Inc.: Hoboken, NJ, USA, 2007; Volume 2. 10. Pole, S. Ayurvedic Medicine: The Principles of Traditional Practice; Jessica Kingsley Publishers: London, UK, 2013. 11. Fang, Z.; Bhandari, B. Effect of spray drying and storage on the stability of bayberry polyphenols. Food Chem. 2011, 129, 1139–1147. [CrossRef][PubMed] 12. Garber, A.J.; Karlsson, F.O. Treatment of dyslipidemia in diabetes. Endocrinol. Metab. Clin. 2001, 30, 999–1010. [CrossRef] 13. Lee, B.R.; Lee, Y.P.; Kim, D.W.; Song, H.Y.; Yoo, K.Y.; Won, M.H.; Kang, T.C.; Lee, K.J.; Kim, K.H.; Joo, J.H.; et al. Amelioration of streptozotocin- induced diabetes by Agrocybe chaxingu polysaccharide. Mol. Cells 2010, 29, 349–354. [CrossRef][PubMed] 14. Hu, S.H.; Wang, J.C.; Lien, J.L.; Liaw, E.T.; Lee, M.Y. Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus. Appl. Microbiol. Biotechnol. 2006, 70, 107–113. [CrossRef] [PubMed] 15. Silva, P.I.; Stringheta, P.C.; Teófilo, R.F.; de Oliveira, I.R.N. Parameter optimization for spray-drying microencapsulation of jaboticaba (Myrciaria jaboticaba) peel extracts using simultaneous analysis of responses. J. Food Eng. 2013, 117, 538–544. [CrossRef] 16. Tonon, R.V.; Brabet, C.; Pallet, D.; Brat, P.; Hubinger, M.D. Physicochemical and morphological characterization of açai (Euterpe oleraceae Mart.) powder produced with different carrier agents. Int. J. Food Sci. Technol. 2009, 44, 1950–1958. [CrossRef] 17. Moreira, G.E.G.; de Azeredo, H.M.C.; de Medeiros, M.d.F.D.; de Brito, E.S.; de Souza, A.C.R. Ascorbic acid and anthocyanin retention during spray drying of acerola pomace extract. J. Food Process. Preserv. 2010, 34, 915–925. [CrossRef] 18. Tonon, R.V.; Brabet, C.; Hubinger, M.D. Influence of process conditions on the physicochemical properties of acai (Euterpe oleraceae Mart.) powder produced by spray drying. J. Food Eng. 2008, 88, 411–418. [CrossRef] 19. Mathiowitz, E. Microencapsulation. In Encyclopedia of controlled drug delivery; Mathiowitz, E., Ed.; John Wiley and Sons: New York, NY, USA, 1999. 20. Mahdavi, S.A.; Jafari, S.M.; Assadpoor, E.; Dehnad, D. Microencapsulation optimization of natural anthocyanins with maltodextrin, gum arabic and gelatin. Int. J. Biol. Macromol. 2016, 85, 379–385. [CrossRef] [PubMed] 21. Varghese, C.P. Antioxidant and anti-inflammatory activity of Eurycoma longifolia Jack, A traditional medicinal plant in Malaysia. Int. J. Pharm. Sci. Nanotechnol. 2013, 5, 1875–1878. 22. Shahzad, M.; Shabbir, A.; Wojcikowski, K.; Wohlmuth, H.; Gobe, G.C. The Antioxidant Effects of Radix Astragali (Astragalus membranaceus and related Species) in Protecting Tissues from Injury and Disease. Curr. Drug Target. 2016, 17, 1331–1340. [CrossRef] 23. Patra, A.; Jha, S.; Murthy, P.N. Phytochemical and pharmacological potential of Hygrophila spinosa T. Anders. Pharmacol. Rev. 2009, 3, 330–341. Nutrients 2018, 10, 843 22 of 25

24. Sharma, G.; Kumar, M. Antioxidant and modulatory role of Chlorophytum borivilianum against arsenic induced testicular impairment. J. Eniron. Sci. 2012, 24, 2159–2165. [CrossRef] 25. Suleria, H.A.R.; Butt, M.S.; Anjum, F.M.; Saeed, F.; Batool, R.; Ahmad, A.N. Aqueous garlic extract and its phytochemical profile; special reference to antioxidant status. Int. J. Food Sci. Nutr. 2012, 63, 431–439. [CrossRef][PubMed] 26. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30. [CrossRef] 27. AOAC. Official Methods of Analysis of the Association of Official Analytical Chemists; AOAC: Washington, DC, USA, 1990. 28. Cano-Chauca, M.; Stringheta, P.C.; Ramos, A.M.; Cal-Vidal, J. Effect of the carriers on the microstructure of mango powder obtained by spray drying and its functional characterization. Innov. Food Sci. Emerg. Technol. 2005, 6, 420–428. [CrossRef] 29. Caparino, O.A.; Tang, J.; Nindo, C.I.; Sablani, S.S.; Powers, J.R.; Fellman, J.K. Effect of drying methods on the physical properties and microstructures of mango (Philippine ‘Carabao’ var.) powder. J. Food Eng. 2012, 111, 135–148. [CrossRef] 30. Young-In, K.; Apostolidis, E.; Shetty, K. Inhibitory potential of wine and tea against α-amylase and α-glucosidase for management of hyperglycemia linked to type 2 diabetes. J. Food Biochem. 2008, 32, 15–31. 31. Apostolidis, E.; Kwon, Y.I.; Shetty, K. Inhibitory potential of herb, fruit, and funga-enriched cheese against key enzymes linked to type 2 diabetes and hypertension. Innov. Food Sci. Emerg. Technol. 2007, 8, 46–54. [CrossRef] 32. Moreda-Pineiro, J.; Herbello-Hermelo, P.; Domínguez-González, R.; Bermejo-Barrera, P.; Moreda-Piñeiro, A. Bioavailability assessment of essential and toxic metals in edible nuts and seeds. Food Chem. 2016, 205, 146–154. [CrossRef][PubMed] 33. OECD. OECD Guidelines for the Testing of Chemicals, Repeated Dose 28-Day Oral Toxicity Study in Rodents; OECD: Paris, France, 1995; Volume 407. 34. Bringmann, G.; Kajahn, I.; Neusüß, C.; Pelzing, M.; Laug, S.; Unger, M.; Holzgrabe, U. Analysis of the glucosinolate pattern of Arabidopsis thaliana seeds by capillary zone electrophoresis coupled to electrospray ionization-mass spectrometry. Electrophoresis 2005, 26, 1513–1522. [CrossRef][PubMed] 35. Golde, P.H.; van der Westelaken, M.; Bouma, B.N.; van de Wiel, A. Characteristics of piraltin, a polyphenol concentrate, produced by freeze-drying of red wine. Life Sci. 2004, 74, 1159–1166. [CrossRef][PubMed] 36. Silva, P.B.; Duarte, C.R.; Barrozo, M.A.S. Dehydration of acerola (Malpighia emarginata D.C.) residue in a new designed rotary dryer: Effect of process variables on main bioactive compounds. Food Bioprod. Process. 2016, 98, 62–70. [CrossRef] 37. Ramírez, M.J.; Giraldo, G.I.; Orrego, C.E. Modeling and stability of polyphenol in spray-dried and freeze-dried fruit encapsulates. Powder Technol. 2015, 277, 89–96. [CrossRef] 38. Burin, V.M.; Rossa, P.N.; Ferreira-Lima, N.E.; Hillmann, M.C.; Boirdignon-Luiz, M.T. Anthocyanins: Optimization of extraction from Cabernet Sauvignon grapes, microencapsulation and stability in soft drink. Int. J. Food Sci. Technol. 2011, 46, 186–193. [CrossRef] 39. Souza, V.B.; Thomazini, M.; de Carvalho Balieiro, J.C.; Fávaro-Trindade, C.S. Effect of spray drying on the physicochemical properties and color stability of the powdered pigment obtained from vinification byproducts of the Bordo grape (Vitis labrusca). Food Bioprod. Process. 2015, 93, 39–50. [CrossRef] 40. Franceschinis, L.; Salvatori, D.M.; Sosa, N.; Schebor, C. Physical and functional properties of blackberry freeze- and spray-dried powders. Dry. Technol. 2014, 32, 197–207. [CrossRef] 41. Ezhilarasi, P.N.; Indrani, D.; Jena, B.S.; Anandharamakrishnan, C. Freeze drying technique for microencapsulation of Garcinia fruit extract and its effect on Bread quality. J. Food Eng. 2013, 117, 513–520. [CrossRef] 42. Gurak, P.D.; Cabral, L.M.C.; Rocha-Leao, M.H. Production of grape juice powder obtained by freeze-drying after concentration by reverse osmosis. Braz. Arch. Biol. Technol. 2013, 56, 1011–1017. [CrossRef] 43. Cortes-Rojas, D.F.; Souza, C.R.F.; Oliveira, W.P. Optimization of spray drying conditions for production of Bidens pilosa L. dried extract. Chem. Eng. Res. Des. 2015, 93, 366–376. [CrossRef] 44. Dag, D.; Kilercioglu, M.; Oztop, M.H. Physical and chemical characteristics of encapsulated goldenberry (Physalis peruviana L.) juice powder. LWT Food Sci. Technol. 2017, 83, 86–94. [CrossRef] Nutrients 2018, 10, 843 23 of 25

45. Çam, M.; Içyer, N.C.; Erdogan, F. Pomegranate peel phenolics: Microencapsulation, storage stability and potential ingredient for functional food development. LWT Food Sci. Technol. 2014, 55, 117–123. [CrossRef] 46. Khazaei, K.M.; Jafari, S.M.; Ghorbani, M.; Kakhki, A.H. Application of maltodextrin and gum Arabic in microencapsulation of saffron petal’s anthocyanins and evaluating their storage stability and color. Carbohydr. Polym. 2014, 105, 57–62. [CrossRef][PubMed] 47. Man, Y.B.C.; Irwandi, J.; Abdullah, W.J.W. Effect of different types of maltodextrin and drying methods on physico-chemical and sensory properties of encapsulated durian flavor. J. Sci. Food Agric. 1999, 79, 1075–1080. [CrossRef] 48. Sawale, P.D.; Patil, G.R.; Hussain, S.A.; Singh, A.K.; Singh, R.R.B. Release Characteristics of Polyphenols from Microencapsulated Terminalia Arjuna Extract: Effects of Simulated Gastric Fluid. Int. J. Food Prop. 2017, 20, 3170–3178. [CrossRef] 49. Adhikari, B.; Howes, T.; Bhandari, B.R.; Troung, V. Effect of addition of maltodextrin on drying kinetics and stickiness of sugar and acid-rich during convective drying: Experiments and modelling. J. Food Eng. 2004, 62, 53–68. [CrossRef] 50. Mitchell, H.L. The role of the bulking agent polydextrose in fat replacement. In Handbook of Fat Replacers; Rollern, S., Jones, S.A., Eds.; CRC Press: Boca Raton, FL, USA, 1996; pp. 235–249. 51. Kapoor, M.P.; Juneja, L.R. Partially hydrolyzed guar gum dietary fiber. In Fiber Ingredients: Food Applications and Health Benefits; Cho, S.S., Samuel, P., Eds.; CRC Press: Boca Raton, FL, USA, 2009; pp. 70–120. 52. Al-Assaf, S.; Philips, G.O.; Williams, P.A. Studies on Acacia exudates gums: Part II. Molecular weight comparison of the Vulgares and Gummiferae series of Acacia gums. Food Hydrocoll. 2005, 19, 661–667. [CrossRef] 53. Kumar, P.S.; Sudha, S. Evaluation of α amylase and α glucosidase inhibitory properties of selected seaweeds from Gulf of Mannar. Int. Res. J. Pharm. 2012, 3, 128–130. 54. Lavelli, V.; Harsha, P.S.; Ferranti, P.; Scarafoni, A.; Iametti, S. Grape skin phenolics as inhibitors of mammalian α-glucosidase and α-amylase effect of food matrix and processing on efficacy. Food Funct. 2016, 7, 1655–1663. [CrossRef][PubMed] 55. Kazeem, M.I.; Ogunbiyi, J.V.; Ashafa, A.O.T. In vitro Studies on the Inhibition of α-Amylase and α- Glucosidase by Leaf Extracts of Picrali manitida (Stapf) Gin H., Rigalleau V. Post-prandial hyperglycemia and Diabetes. Diabete Metab. 2000, 26, 265–272. 56. Sima, A.A.; Chakrabarti, S. Long-term suppression of postprandial hyperglycaemia with acarbose retards the development of neuropathies in the BB/W-rat. Diabetologia 1992, 35, 325–330. [CrossRef][PubMed] 57. Kwon, Y.I.; Apostolidis, E.; Shetty, K. Evaluation of pepper (Capsicum annuum) for management of diabetes and hypertension. J. Food Biochem. 2007, 31, 370–385. [CrossRef] 58. Sultan, M.T.; Buttxs, M.S.; Qayyum, M.M.N.; Suleria, H.A.R. Immunity: Plants as effective mediators. Crit. Rev. Food Sci. Nutr. 2014, 54, 1298–1308. [CrossRef][PubMed] 59. Kim, J.S.; Kwon, C.S.; Son, K.H. Inhibition of α-glucosidase and amylase by Luteolin, a flavonoid. Biosci. Biotechnol. Biochem. 2000, 64, 2458–2461. [CrossRef][PubMed] 60. Matsui, T.; Ogunwande, I.A.; Abesundara, K.J.M.; Sumoto, K.M. Anti-hyperglycemicpotential of natural products. Mini Rev. Med. Chem. 2006, 6, 349–356. [CrossRef][PubMed] 61. Matsuda, H.; Morikawa, T.; Yoshikawa, M. Antidiabetogenic constituents from several natural medicines. Pure Appl. Chem. 2002, 74, 1301–1308. [CrossRef] 62. Ogunwande, I.A.; Matsui, T.; Fujise, T.; Matsumoto, K. α-Glucosidase inhibitory profile of Nigerian medicinal plants in immobilized assay system. Food Sci. Technol. Res. 2007, 13, 169–172. [CrossRef] 63. Nunes, G.L.; Boaventura, B.C.B.; Pinto, S.S.; Verruck, S.; Murakami, F.S.; Prudêncio, E.S.; Amboni, R.D.D.M.C. Microencapsulation of freeze concentrated Ilex paraguariensis extract by spray drying. J. Food Eng. 2015, 151, 60–68. [CrossRef] 64. Kuck, L.S.; Norena, C.P.Z. Microencapsulation of grape (Vitis labrusca var. Bordo) skin phenolic extract using gum arabic, polydextrose, and partially hydrolyzed guar gum as encapsulating agents. Food Chem. 2016, 194, 569–576. [CrossRef][PubMed] 65. Kuang, S.S.; Oliveira, J.C.; Crean, A.M. Microencapsulation as a tool for incorporating bioactive ingredients into food. Crit. Rev. Food Sci. Nutr. 2010, 50, 951–968. [CrossRef][PubMed] 66. Chen, C.; Chi, Y.J.; Xu, W. Comparisons on the functional properties and antioxidant activity of spray-dried and freeze-dried egg white protein hydrolysate. Food Bioproess Technol. 2012, 5, 2342–2352. [CrossRef] Nutrients 2018, 10, 843 24 of 25

67. Saikia, S.; Mahnot, N.K.; Mahanta, C.L. Optimization of phenolic extraction from Averrhoa carambola pomace by response surface methodology and its microencapsulation by spray and freeze drying. Food Chem. 2015, 171, 144–152. [CrossRef][PubMed] 68. Eldeen, A.; Kawashty, S.A.; Ibrahim, L.F.; Shabana, M.M.; El-Negoumy, S.I. Evaluation of antioxidant, anti-inflammatory, and antinociceptive properties of aerial parts of Vicia sativa and its flavonoids. J. Nat. Remed. 2004, 4, 81–96. 69. Tisserant, L.P.; Hubert, J.; Lequart, M.; Borie, N.; Maurin, N.; Pilard, S.; Jeande, P.; Aziz, A.; Renault, J.-H.; Nuzillard, J.-M.; et al. 13C NMR and LC-MS Profiling of Stilbenes from Elicited Grapevine Hairy Root Cultures. J. Nat. Prod. 2016, 79, 2846–2855. [CrossRef][PubMed] 70. Chobot, V.; Hadacek, F.; Bachmann, G.; Weckwerth, W.; Kubicova, L. Pro- and Antioxidant Activity of Three Selected Flavan Type Flavonoids: Catechin, Eriodictyol and Taxifolin. Int. J. Mol. Sci. 2016, 17, 1986–1995. [CrossRef][PubMed] 71. Hassan, S.M.; Khalaf, M.M.; Sadek, S.A.; Abo-Youssef, A.M. Protective effects of apigenin and myricetin against cisplatin-induced nephrotoxicity in mice. Pharm. Biol. 2017, 551, 766–774. [CrossRef][PubMed] 72. Cuendet, M.; Potterat, O.; Hostettmann, K. Flavonoids and phenylpropanoid derivatives from Campanula barbata. Phytochemistry 2001, 56, 631–636. [CrossRef] 73. Tang, L.; Ling, A.P.; Koh, R.Y.; Chye, S.M.; Voon, K.G. Screening of anti-dengue activity in methanolic extracts of medicinal plants. BMC Complement. Altern. Med. 2008, 12, 1186–1196. [CrossRef][PubMed] 74. Khaomek, P.; Ichino, C.; Ishiyama, A.; Sekiguchi, H.; Namatame, M.; Ruangrungsi, N.; Saifah, E.; Kiyohara, H.; Otoguro, K.; Omura, S.; et al. In vitro antimalarial activity of prenylated flavonoids from Erythrina fusca. J. Nat. Med. 2008, 62, 217–220. [CrossRef][PubMed] 75. Saeed, F.; Ahmad, R.S.; Arshad, M.U.; Niaz, B.; Batool, R.; Naz, R.; Suleria, H.A.R. Propolis to Curb Lifestyle Related Disorders: An Overview. Int. J. Food Prop. 2015, 18, 420–437. [CrossRef] 76. Agostino, M.; De Simone, F.; Piacente, S.; Pizza, C.; Senatore, F. Quercetagetin 6-O-β-D-glucopyranoside from Tagetes mandonii. Phytochemistry 1997, 45, 201–202. [CrossRef] 77. Zhang, Y.; Wang, C.; Yang, Q.; Jin, Y.; Meng, Q.; Liu, Q.; Dai, Y.; Liu, Z.; Liu, K.; Sun, H. Catalpol attenuates oxidative stress and promotes autophagy in TNF-a-exposed HAECs by up-regulating AMPK. RSC Adv. 2017, 7, 52561–52567. [CrossRef] 78. Aceves, L.; Dublán-García, O.; López-Martínez, L.X.; Novoa-Luna, K.A.; Islas-Flores, H.; Galar-Martínez, M.; García-Medina, S.; Hernández-Navarro, M.D.; Gómez-Oliván, L.M. Reduction of the Oxidative Stress Status Using Steviol Glycosides in a Fish Model (Cyprinus carpio). BioMed Res. Int. 2017, 9, 235–294. 79. Shmuel, Y. Dictionary of Food Compounds with CD-ROM: Additives, Flavors, and Ingredients; Chapman & Hall/CRC: Boca Raton, FL, USA, 2004. 80. Cedrón, J.C.; Gutiérrez, D.; Flores, N.; Ravelo, Á.G.; Estévez-Braun, A. Preparation and antimalarial activity of semisynthetic lycorenine derivatives. Euro. J. Med. Chem. 2013, 63, 722–730. [CrossRef][PubMed] 81. Buckingham, J.; Munasingh, V.R.N. Dictionary of Flavonoids, 1st ed.; CD-ROM: London, UK, 2007. 82. Palluotto, F.; Sosic, A.; Pinato, O.; Zoidis, G.; Catto, M.; Sissi, C.; Catto, B.; Carotti, A. Quinolino [3, 4-b] quinoxalines and pyridazino [4, 3-c] quinoline derivatives: Synthesis, inhibition of topoisomerase II α, G-quadruplex binding and cytotoxic properties. Eur. J. Med. Chem. 2016, 123, 704–717. [CrossRef][PubMed] 83. Fiedor, J.; Brrda, K. Potential Role of Carotenoids as Antioxidants in Human Health and Disease. Nutrients 2014, 6, 466–488. [CrossRef][PubMed] 84. Buckingham, J.; Baggaley, K.H.; Roberts, A.D.; Szabo, L.F. Dictionary of Alkaloids, Second Edition with CD-ROM, 2010. Available online: https://books.google.com/books?id=mynNBQAAQBAJ (accessed on 2 March 2018). 85. Mensah, A.Y.; Houghton, P.J.; Bloomfield, S.; Vlietinck, A.; Vanden Berghe, D. Known and novel terpenes from Buddleja globosa displaying selective antifungal activity against dermatophytes. J. Nat. Prod. 2000, 63, 1210–1213. [CrossRef][PubMed] 86. Chun, H.; Yuan, Y.V.; Kitts, D.D. Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro. Food Chem. Toxicol. 2007, 45, 2219–2227. 87. Wangteeraprasert, R.; Lipipun, V.; Gunaratnam, M.; Neidle, S.; Gibbons, S.; Likhitwitayawuid, K. Bioactive compounds from Carissa spinarum. Phytother. Res. 2012, 26, 1496–1499. [CrossRef][PubMed] Nutrients 2018, 10, 843 25 of 25

88. Stangl, V.; Lorenz, M.; Ludwig, A.; Grimbo, N.; Guether, C.; Sanad, W.; Ziemer, S.; Martus, P.; Baumann, G.; Stangl, K. The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets. J. Nutr. 2005, 135, 172–178. [CrossRef][PubMed] 89. Sumiyoshi, M.; Taniguchi, M.; Baba, K.; Kimura, Y. Antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin isolated from Angelica keiskei roots through the inhibited activation and differentiation of M2 macrophages. Phytomedicine 2015, 22, 759–767. [CrossRef][PubMed] 90. Cui, L.; Decker, E.A. Phospholipids in foods: Pro-oxidants or antioxidants. J. Sci. Food Agric. 2016, 96, 18–31. [CrossRef][PubMed] 91. Dugasani, S.; Pichika, M.R.; Nadarajah, V.D.; Balijepalli, M.K.; Tandra, S.; Korlakunta, J.N. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. J. Ethnopharmacol. 2010, 127, 515–520. [CrossRef][PubMed] 92. Park, M.T.; Kang, J.A.; Choi, J.A.; Kang, C.M.; Kim, T.H.; Bae, S.; Kang, S.; Kim, S.; Choi, W.; Cho, C.-K.; et al. Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells. Clin. Cancer Res. 2003, 9, 878–885. [PubMed] 93. Tezuka, Y.; Gewali, M.B.; Ali, M.S.; Banskota, A.H.; Kadota, S. Eleven Novel Diarylheptanoids and Two unusual Diarylheptanoid Derivatives from the Seeds of Alpinia blepharocalyx. J. Nat. Prod. 2001, 64, 208–213. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).